Basement membranes are the specialized extracellular matrices which interface with both alveolar epithelial and capillary endothelial cells in the lung. Current data indicate these structures contain three types of macromolecules - type IV collagen, laminin and proteoglycans. This type of extracellular maxtrix (ECM) functions not only as a cellular supporting element and a molecular filter, but recent evidence suggests the components of the matrix may influence cell growth and differentiation. As these structures are critical to the normal maintenance of lung function, knowledge about the regulation of these ECM components is essential for understanding lung development and response of the tissue during disease. It is the long-term objective of the proposed investigations to delineate the mechanisms and factors which control the expression of the basement membrane components and the mechanisms through which the components influence cellular function. It is felt that these objectives can be achieved by combining in vitro studies with recombinant DNA techniques to dissect at the molecular level the regulation of ECM biosynthesis in the lung. Recent work in this laboratory has established that cultured fetal rat lung cells (clone 2G3) offer a unique opportunity to investigate in vitro the biosynthesis of ECM components. Thus the first major objective of this proposal has two specific aims: 1) to determine if genetic types of collagen other than type IV are synthesized by the cells; and 2) to assess the effects of EGF and retinoic acid on this biosynthetic parameter. The second major approach to be used for acquiring insight into the regulation of ECM biosynthesis in pulmonary systems will be to construct cloned DNA sequences complimentary to the Alphal(IV) procollagen mRNA isolated from cultured ED-PYS cells for the purpose of creating probes for this rate-limiting element in the biosynthesis of type IV collagen, and this major objective has four specific aims: 1) to construct cloned cDNA sequences in bacteria against ED-PYS mRNA; 2) to identify those recombinant molecules containing sequences complimentary to the Alphal(IV) procollagen mRNA; 3) to use these clones to identify and isolate the gene coding for this ECM component; and 4) to develop the experimental methodologies using the cloned cDNA sequences to quantitate and measure changes in Alphal(IV) procollagen mRNA levels in vivo. It is felt that this combination of approaches will establish a legitimate experimental milieu for future investigations aimed at elucidating the mechanisms that regulate extracellular matric biosynthesis in the lung.